A total of 29 differentially expressed inflammatory factors were detected between the EVs secreted by BMECs from the Icariin-treated group and the Model group. The EVs secreted by BMECs could improve cell viability, decrease cell apoptosis, and promote cell migration and angiogenesis under the intervention of glucocorticoids. Meanwhile, icariin intervention could reinforce these protective effects of BMECs-derived EVs. To sum up, the present study indicates that icariin acts as a promising candidate for treating glucocorticoid-induced injury of BMECs and bone diseases, partially through the autocrine activity of EVs. In vivo or animal studies are still required to better understand the function of BMECs-derived EVs.Glucocorticoids could induce injury and apoptosis of bone microvascular endothelial cells (BMECs) in the femoral head, which is associated with the development of osteonecrosis and osteoporosis. Icariin is a prenylated flavonol glycoside isolated from Epimedium brevicornum, serving as the main active pharmaceutical constituent to treat bone loss. Currently, the impact of the autocrine activity of extracellular vesicles (EVs) induced by icariin on the glucocorticoid-induced injury of BMECs is still to be confirmed. In this study, EVs were isolated from BMECs treated with and without icariin by super-speed centrifugation. Although icariin treatment would not significantly change the size and total protein content of BMECs-derived EVs, expression of EVs-carried vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGF-β1) was enhanced and numerous miRNAs involved in cell proliferation and apoptosis were upregulated  or downregulated  ( Synthetic glucocorticoids (GCs)  are a class of anti-inflammatory and immunosuppressive agents widely prescribed in medical conditions, such as rheumatoid arthritis, bronchial asthma, severe acute respiratory syndrome (SARS), systemic lupus erythematosus, and acute urticaria . GlucocoIn addition to damaging bone tissues, glucocorticoids have shown a toxic effect on bone microvascular networks . The bonIcariin (ICA/C33H40O15)  is the mThe updated Guidelines of the International Society for Extracellular Vesicles (ISEV) on the minimal information for studies of extracellular vesicles (MISEV) provided recommendations on the experimental methods and minimal information required in the reporting and nomenclature of extracellular vesicles (EVs), a population of phospholipid bilayer-enclosed particles ranging from 20 nanometers to 10 microns or more in diameter . The EVsIn this study, we aimed to investigate the role of the autocrine activity of ECs-derived EVs in the development of glucocorticoid-induced injury of BMECs and icariin intervention. Fractures associated with glucocorticoid-induced osteoporosis usually occur in regions with massive cancellous bone, especially the spine and proximal femur; meanwhile, osteonecrosis predominantly arises in the femoral head . Given tThis experimental protocol was designed following the World Medical Association Declaration of Helsinki on ethical principles for medical research involving human subjects and was approved by the Ethics Committee of China-Japan Friendship Hospital. The inclusion criteria were patients diagnosed as primary osteoarthritis (OA) of the hip, osteoarthritis secondary to developmental dysplasia of the hip (DDH), as well as femoral neck fracture, and subjected to total hip arthroplasty. Written informed consent must be signed before surgery. Eventually, BMEC isolated from a total of eighteen donors  admitted to the orthopedic department of China-Japan Friendship Hospital from May 2017 to January 2021 were used to conduct the experiment presented herein.The BMECs were isolated following the methods described in the previous article . The nor3/well, cultured in a complete medium for 12 h, and treated with icariin  in different concentrations  for another 24 h. Icariin was dissolved in DMSO, whose final concentration in the organ bath solution did not exceed 0.1% and therefore would not cause a significant effect on the survival and proliferation of endothelial cells [The BMECs were seeded in 96-well plates at 5 × 10al cells . Then, Bg for 18 min) with and without icariin for 24 h. Then, the supernatant was collected to isolate EVs using the exosome isolation kit . Each group of EVs was isolated from an equivalent number of BMECs and resuspended in PBS. The diameter distribution and volume of extracted EVs were analyzed using nanoparticle tracking analysis  and the morphology was observed by transmission electron microscope . The EVs-carried proteins were assessed by Western blot assay using primary antibodies to CD9  and CD81 . After adherence, BMECs were cultured in an EVs-free medium | > 1 and p-value ≤ 0.05 as the threshold. Then, genes related to proliferation, angiogenesis and apoptosis of endothelial cells were selected for confirmation by using qPCR. The reverse transcription process was performed using the One-Step PrimeScript® miRNA cDNA Synthesis Kit  and the amplification reaction was conducted using the SYBR® Premix Ex TaqTM II kit . The primer sequences of investigated miRNAs were: hsa-miR-1469, forward, 5′-GCGCAGCTGGTAAAATGGAA-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; hsa-miR-133a-5p, forward, 5′-GCCTCGGCGCGGGGCGCG-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′; hsa-miR-10b-5p, forward, 5′- GCTACCCTGTAGAACCGAA-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′.After extracting RNA from EVs by using TRIzol reagent, high-throughput sequencing of EVs-carried miRNAs was performed, according to standard procedures provided by Illumina. A small RNA sequencing library was prepared using the TruSeq Small RNA Sample Prep Kits . Then, the constructed library was sequenced using Illumina Hiseq 2000/2500 platform. MiRNAs with lower expression  were first removed from the analysis. Then, differentially expressed genes between BMECs in the icariin-treatment and the non-treatment groups were screened using |logTotal proteins were extracted from EVs and measured using a BCA protein assay kit . A total of 200 μL of BCA working solution was added to 10 µL of extracted protein sample per well of 96-well plates and incubated in the dark for 30 min at 37 °C. The OD value was measured at 750 nm, and the protein concentration was calculated from the standard curve, with bovine serum albumin as the standard protein.p-values were calculated and converted to adjusted p-values  for comparisons by false discovery rate correction of the Benjamini and Hochberg test. A cutoff point of adj. p. val < 0.05 and |fold change (FC)| > 1.2 were used to selected DEPs. Then, the PCA plot, heatmap, and volcano plot of the DEPs were drawn using the ggplot2 package in R software. The expression levels of specific protein of interest were determined by ELISA.The isolated proteins were scanned by using the GSH-CAA-440 array to obtain the raw data. Then, the Raybiotech software was used to remove the chip background and normalize the original data. Differentially expressed proteins (DEPs) between different groups were identified using the limma package (Version 3.42.2) in R software . Individual https://david-d.ncifcrf.gov/ (accessed on 1st January 2022)), for enrichment analysis, including gene ontology (GO) and Kyoto Encyclopedia of Gene and Genome (KEGG) pathways. The obtained results were visualized in the R ggplot2 package and a p-value of <0.05 was considered statistically significant.The DEPs were uploaded to an online bioinformatics database, the Database for Annotation, Visualization, and Integrated Discovery (DAVID) version 6.8 Beta (http://string-db.org (accessed on 1st January 2022)) [https://www.networkanalyst.ca/faces/home.xhtml (accessed on 1st January 2022)).The protein–protein interaction (PPI) network was constructed by using the multiple protein online tool in the STRING database )  and visuAccording to the intervention condition, BMECs were divided into three groups: in the Icariin-BMEC-EVs group, BMECs were cultured in the presence of EVs derived from BMECs treated with icariin; in the BMEC-EVs group, BMECs were co-cultured with EVs isolated from BMECs without icariin treatment, and in the Model group, BMECs were free from EVs’ intervention. In the former two groups, the EVs’ concentration was adjusted to 200 ng/uL. After 24 h of cell culture, all of the cells in the three groups were treated with hydrocortisone.The BMECs were inoculated in 6-well plates and when the cells reached confluence as a monolayer, a 1 mL pipette tip was used to gently scratch the cell layer following the center lines of wells. An EVs and hydrocortisone (0.3 mg/mL) intervention was performed following the steps described, before and after 12 h and 24 h, images were taken under an inverted microscope.The BMECs, with and without EVs intervention, were inoculated to 24-well plates coated with enriched growth factor Matrigel  for 24 h and then treated with hydrocortisone (0.06 mg/mL). After 4 and 8 h of hydrocortisone intervention, images were taken under the inverted microscope .After 12 h of hydrocortisone (0.3 mg/mL) intervention, BMECs were harvested and washed twice with PBS at 4 °C and resuspended with 300 µL of binding buffer. A total of 100 μL of cell suspension, 5 μL of AnnexinV-FITC, and 10 μL of propidium iodide (PI) solution (20 ng/L) were mixed at room temperature, incubated in the dark for 15 min, and then the fluorescence intensity was detected by using a flow cytometer.t-test and one-way analysis of variance (ANOVA). A p-value < 0.05 indicates a statistically significant difference. All statistical tests were performed using GraphPad Prism statistical software .Data are presented in the form of mean ± standard deviation (SD). The differences between groups were analyzed by the Student’s The normal cancellous bone from the resected femoral head A was pro−6 M, 1 × 10−5 M, 5 × 10−5 M, and 1 × 10−4 M groups, respectively) . The proliferation promotion effects of icariin in the 5 × 10−5 M and 1 × 10−4 M groups were significantly better in comparison with the other groups . Meanwhile, no significant difference was identified between these two groups . So, 5 × 10−5 M was chosen as the optimal concentration for subsequent experiments.Compared with the icariin-free group, the proliferation rate of BMECs increased as the concentration of icariin increased  A. There p < 0.01). The control group had a relatively low concentration of EVs (9.85 ± 0.47 × 106/mL). After being treated with icariin, the concentration of EVs increased to 1.54 ± 0.08 × 107/mL. The BCA results showed that the protein content of conditioned media was 132.63 ± 3.33 μg/mL in the control group and 138.56 ± 9.58 μg/mL in the icariin group. The difference in protein content of conditioned media between the two groups was not statistically significant (p < 0.01). CD9 and CD81, two representative EVs surface markers, were highly expressed in EVs isolated from cells of both the control group and the icariin group (p < 0.01) , the average diameter of isolated EVs was 86.68 ± 1.97 nm, while in the icariin group, the average diameter of EVs was 85.35 ± 1.65 nm. We found that icariin treatment did not significantly alter the diameter of EVs derived from BMEC cells in the two groups  B. By TEM < 0.01) C.p < 0.01)  D.p < 0.01)  to confirm biological variability between different EVs samples A. In tot < 0.01) .GO enrichment analysis of 29 DEPs between EVs isolated from the Icariin-treated group and the Model group was performed to identify the most relevant biological processes (BPs), molecular functions (MFs), and cellular components (CCs). The top ten enriched terms in BPs, CCs, and MFs are presented in The interactions between the 29 DEPs detected between EVs isolated from the Icariin-treated and the Model groups, which consisted of 24 nodes and 35 edges A, were cThe TF-miRNA interacted network was constructed through network analysis of 29 DEPs in Cytoscape B, which p < 0.05) (p < 0.05). According to the tube formation assay, the ability to promote migration and angiogenesis of microvascular endothelial cells was more pronounced in the Icariin-BMEC-EVs group in comparison with the BMEC-EVs group (p < 0.05), leading to a greater number of branch points, cavities, and longer tube length  A,B. EVs e length C–F.p-value < 0.01). This effect was dominant in the Icariin-BMEC-EVs group compared with the BMEC-EVs group (p < 0.01) (After treatment with hydrocortisone (0.3 mg/mL) for 0, 1, 2, 3, and 4 days, the viability of BMECs in the BMEC-EVs group and the Icariin-BMEC-EVs group was significantly enhanced in comparison with that of the Model group ( < 0.01) A,B.p-value < 0.01). Similarly, the inhibitory effect for apoptosis was more significant in the Icariin-BMEC-EVs group compared with the BMEC-EVs group   and the Icariin-BMEC-EVs group (10.70 ± 0.7%) were lower than those in the Model group (35.67 ± 1.99%) ( < 0.01) C. A redu−6 M to 5 × 10−5 M [−5 M was identified as the optimal concentration for promoting BMECs’ viability and reinforcing the protective effect of autocrine EVs for glucocorticoid-induced injury of BMECs. This effect was partially realized by changing the protein and RNA profiling in secreted EVs of bone microvascular endothelial cells.By using bone microvascular endothelial cells isolated from the femoral head, the experimental conditions are closer to the microvasculature in bone than those using human umbilical vein endothelial cells. The preceding finding has revealed that a hydrocortisone concentration of more than 0.3 mg/mL would induce typical proliferation inhibition and apoptosis of BMECs . In this× 10−5 M . The preThe BMECs-derived EVs, defined as EVs released by BMECs during external activation, might be initiated by chemical  and physical  factors . The resECs-derived EVs affects recipient cells’ function and modulates key events in the development of multiple disorders; the effect could be either beneficial or detrimental. It was also shown that capillary endothelial tip cells secrete EVs incorporating Delta-like 4 ligands, which are passed to the tip cells of the endothelial sprout, promote cell motility, and suppress their proliferation . SenesceBy constructing a TF-miRNA-DEP interacted network, we selected multiple important miRNAs  and transcription factors  that might be involved in the dysregulation of these proteins. In former studies, these molecules were demonstrated to be vital for vascular development and angiogenesis ,36. MYC This study is not without limitations. First, extrapolation of the results from cell models to clinical practice should be performed with caution. It is needed to consider the impact of factors in vivo. Second, although hsa-miR-133a-5p and hsa-miR-10b-5p were enriched after icariin intervention, we did not perform a further experiment to test whether they could take effect on the reversion of glucocorticoid-induced cellular injury. Last but not least, the interaction of EVs secreted by BMECs and other cell types needs to be further investigated. It is well-acknowledged that icariin could affect multiple cell types existing in bones, for example, MSCs and osteocytes; meanwhile, EVs can modulate inflammation, regulate the activation and migration of monocytes, and affect the differentiation of MSCs. Therefore, it is rational to assume that other kinds of cells in the bone or even cells of cardiovascular systems in the distance may also engulf BMECs-derived EVs may also be engulfed by different types of cells. In vivo and animal studies are required to confirm these findings and further illuminate the action mechanism of identified miRNAs and proteins for the protective effects of icariin to glucocorticoid-induced injury of the BMECs.The present study indicates that the autocrine activity of extracellular vesicles could significantly improve glucocorticoid-induced injury of BMECs. Icariin intervention could reinforce these effects and may act as a promising candidate for glucocorticoid-induced bone diseases. In vivo or animal studies are still required to gain a better understanding of the function of BMEC-derived extracellular vesicles.


Antrodia camphorata is rich in a variety of bioactive ingredients; however, the utilization efficiency of the residue of A. camphorata is low, resulting in serious waste. It is necessary to deeply study the functional components of A. camphorata residues to achieve high-value utilization. In this study, the components, structural characteristics, and functional properties of alkali-extracted dietary fiber extracted from residues of A. camphorata  were investigated. There were similar components and structural characteristics of ACA-DK (extract from basswood cultured) and ACA-DF (extract from dish cultured). The two alkali-extracted dietary fiber were composed of mainly cellulose and xylan. However, ACA-DK has better adsorption capacities than ACA-DF on lipophilic substances such as oil (12.09 g/g), cholesterol (20.99 mg/g), and bile salts (69.68 mg/g). In vitro immunomodulatory assays stated that ACA-DK had a good effect on promoting the proliferation of RAW 264.7 cells and can activate cell phagocytosis, NO synthesis, and other immune capabilities. The edible fungus A. camphorata is a good source of functional dietary fiber. The alkali-extracted dietary fiber of A. camphorata might be used as a functional ingredient in the medicine and food industry.  However, the functions of dietary fiber from different sources are quite different. Dietary fiber extracted from grain has a protective effect on T2DM, while fruit fiber has almost no protective effect . Macrophage RAW 264.7 is a good in vitro experimental platform to measure the immunomodulatory or the anti-inflammatory activity of polysaccharides  and stored in the refrigerator at 4°C. The residues were extraction by hot water (95°C for 2 h) before experiments, to remove water-soluble polysaccharides. All the monosaccharide standards, MTT, lipopolysaccharide (LPS), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Co. . High-glucose DMEM was purchased from Wisten Biotechnology Co. Ltd. . A mouse IL-6 ELISA kit  and a TNF-α ELISA kit  were purchased from R&D Systems China Co. Ltd. . All other reagents used in this study were of analytical grade.In this experiment, the residues of Antrodia camphorata residues were washed with ethanol and distilled water. Then, the residues were dried at 60°C and ground to a powder (filtered by a 60-mesh sieve). The powder was packaged and stored at −20°C until further analysis.A. camphorata residues using the described method with some modifications , followed by centrifugated at 8,000 rpm for 15 min at 4°C. The supernatants were dialyzed for 3 d and freeze drying at −80°C for 72 h. The dietary fiber extracted from the fruiting body of A. camphorata was coded as ACA-DK. The dietary fiber extracted from dish-cultured hypha of A. camphorata was coded as ACA-DF.The dietary fiber were extracted from The molecular weights of dietary fiber were determined by using an HPLC system , with DMSO as the mobile phase at a flow rate of 1 mL/min at 35°C. Molecular weights of dietary fiber were calculated by the retention time.Total sugars were estimated by the phenol–sulfuric acid method  and C: NaOAc (1 mol/L). The elution condition started A:B:C = 92:8:0; 0-20 min, linear gradient to A:B:C =87:8:5; 20-30 min, linear gradient to A:B:C = 78:8:20; 30-50 min, linear gradient to A:B:C = 20:80:0.Monosaccharide composition of dietary fiber were measured by HPAEC-PAD (Dionex ICS 3000)  were well ground and blended with KBr (9 mg) and then pressed into a 1 mm pellet for FT-IR analysis  in the resolution range from 400 to 4,000 cmCrystalline structural analysis of the freeze-dried samples was performed by using an X-ray diffractometer (MiniFlex600) with a Cu-Kα radiation source (λ = 1.542 Å) at a voltage of 40 kV and an incident current of 40 mA  containing water and m1 is the weight of the dry sample (g).where mWSC was determined by the method reported by Sowbhagya et al.  with app2 is the volume of the hydrated sample, V0 is the volume of the sample prior to hydration, and M0 is the weight of the sample prior to hydration.where VOil adsorption capacity was measured according to the method of Luo et al. . The sam3 is the weight of the residue (g) containing oil and M1 is the weight of the dry sample (g).where MCholesterol binding capacity of the samples were investigated according to the procedure described by previously reported with slight modification  were seeded in 96-well culture plates under conditions  for 24 h. The DEME mediums containing different concentrations of ACA-DK and ACA-DF  were added into each well and incubated with the cells for 24 h. LPS (1 μg/mL) was used as the positive control group. After incubation, the medium was removed, and cells were incubated with 100 μL of MTT (0.5 mg/mL) solutions for 4 h. Next, the MTT solution was discarded, and dimethyl sulfoxide (100 μL of DMSO) was added and shaken for 10 min until no visible particulate matter detected. Finally, the absorbance was measured at 570 nm, and the cell proliferation ratio was determined. The morphology of the RAW 264.7 cells was observed via an inverted fluorescence microscope.Cell proliferation was quantified by a standard MTT assay  were seeded in 96-well culture plates under conditions  for 24 h. The DEME medium containing different concentrations of ACA-DK and ACA-DF  was added into each well and incubated with the cells for 24 h. LPS (1 μg/mL) was used as the positive control group. After incubation, the medium was removed, and the cells were incubated with neutral red  solutions for 30 min. Next, the supernatant was discarded, and the cells were washed with PBS (200 μL per well) for three times at room temperature to remove the redundant neutral red. Then, a 100 μL of the lysis solution contained an equal volume ratio of ethanol , and acetic acid  was added into the well and lysed at 4°C for 12 h. Finally, the absorbance was measured at 540 nm, and the phagocytic activity was determined.The promoting effects of ACA-DK and ACA-DF on phagocytosis of RAW 264.7 cells were determined by neutral red uptake assay  were seeded in 96-well culture plates under conditions  for 24 h. The RAW 264.7 cells were treated for 24 h with different concentrations of ACA-DK and ACA-DF . LPS (1 μg/mL) was used as the positive control group. After incubation, the supernatant was collected from cultures and mixed with an equal volume of Griess reagent and incubated for 30 min at room temperature. Nitrite production was determined by measuring absorbance at 540 nm with NaNO2 as a standard.RAW 264.7 cells  were seeded in 96-well culture plates under conditions  for 24 h. The RAW 264.7 cells were treated for 24 h with different concentration of ACA-DK and ACA-DF . LPS (1 μg/mL) was used as the positive control group. After incubation, the supernatant was collected from cultures for analysis of TNF-α and IL-1β using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions.RAW 264.7 cells  was used to assess the differences between mean values. Statistical analysis was performed with ANOVA (SPSS 24.0). A A. camphorata from basswood-cultivated and dish-cultured extract used in this experiment were the residues of ethanol and hot water extraction. As shown in The samples of The monosaccharide composition of dietary fiber has an important influence on its properties. As shown in The morphology and structure of the ACA-DK and ACA-DF were analyzed by scanning electron microscope (SEM), and the results are shown in −1, which reflects functional groups of dietary fiber from A. camphorata. Similar typical peaks were observed from the spectral of ACA-DK and ACA-DF. As shown in −1 for O-H group vibration, mainly of the native hemicelluloses  stretching vibration of hemicelluloses, indicating a dominant xylan of the alkali-soluble hemicelluloses , water holding capacity (WHC), and oil adsorption capacity (OAC) were presented and compared in P < 0.05). While the cell viability of ACA-DK was significantly higher than that of ACA-DF, when the concentration was up to 100 μg/mL. Neutral red uptake was carried out to explore the effects of ACA-DK and ACA-DF on the phagocytic activity of LPS-treated RAW 264.7 macrophages cells. As shown in P < 0.01). The two dietary fiber samples could both enhance the phagocytic ability of RAW 264.7 cell significantly. However, the phagocytic uptake ability of the RAW 264.7 cells was promoted significantly and presented in a good concentration-dependent manner after treated with ACA-DK. It was noted that the phagocytic ability of RAW 264.7 cells with a high concentration of ACA-DK was significantly lower than that of the LPS-treated group. The results suggest that ACA-DK is superior to ACA-DF in the activation of phagocytosis.To analyze whether ACA-DK and ACA-DF cause toxicity on RAW 264.7 macrophages, the cell viability was measured by MTT assay. As shown in P < 0.01). The production of NO was significantly promoted by ACA-DK and ACA-DF in a time- and concentration-dependent manner (P < 0.01), and the two dietary fiber had the similar ability of promoting NO production.Effects of ACA-DK and ACA-DF on NO production are presented in The effects of each group on RAW 264.7 cell morphology was observed under an inverted microscope. As shown in P < 0.01). Stimulatory activity of ACA-DK and ACA-DF on macrophages for 24 h caused an increase in the production of TNF-α and IL-1β cytokines in a dose-dependent manner. ACA-DK could significantly promote the release of TNF-α and IL-1β cytokines (P < 0.01) compared to ACA-DF in the concentration range of 50–500 μg/mL. After 24 h of the ACA-DK treatment, the secretion of TNF-α and IL-1β reached the highest levels for 484.43 ± 18.77 pg/mL and 60.39 ± 3.47 ng/mL, respectively.To further investigate the immunomodulatory effects of ACA-DK and ACA-DF on macrophage cells, pro-inflammatory cytokines, such as TNF-α and IL-1β, were determined using ELISA kits. As shown in Antrodia camphorata is a medicinal fungus with a variety of bioactivities. At present, most of A. camphorata is treated with solvents to extract small molecular bioactive components and polysaccharides. However, the residue after extraction has not been effectively developed, and the waste of resources is more serious. In this study, the dietary fiber of A. camphorata residue was obtained by alkaline extraction, and its adsorption properties and immune regulation properties were studied.Dietary fiber is widely found in grains, fruits, mushrooms, and other foods and has good biological activity. Although dietary fiber is hardly degraded in the intestine, its good adsorption and looseness could promote intestinal peristalsis and promote the excretion of dietary cholesterol and fat from the body. A. camphorata is composed of mainly cellulose and xylan. There are significant differences in the structure of the dietary fiber extract from common foods. The monosaccharide composition of ACA-DK and ACA-DF is significantly different from dietary fiber derived from grains and fruits , the Shanghai Agriculture Applied Technology Development Program (Grant Number 2019-02-08-00-07-F01152), and the Shanghai Engineering Research Center of Food Microbiology Program (Grant Number 19DZ2281100).JL was employed by Honest and Humble Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


H2 cytokine response in epithelial cells, is an important step in developing the inflammatory response of the nasal mucosa. In this study, we investigated whether SST may ameliorate the TDI-induced AR-related symptoms in rats and inhibit IL-33 release from nasal epithelial cells. An AR rat model was generated by sensitization and induction with TDI. SST was administered during the sensitization period. AR-related symptoms in rats were evaluated, and IL-33 release was measured both in vivo and in vitro. SST suppressed symptoms appearing in TDI-induced AR model rats, such as elevated serum histamine and IL-33 levels in nasal lavage fluid (NLF)/serum, which were suppressed by SST administration. TDI-induced IL-33 release from the nasal epithelial cell nuclei was also observed and suppressed in SST-treated rats and cultured nasal epithelial cells. These results suggest that SST ameliorates the symptoms of TDI-induced AR at least partially by inhibiting IL-33 release from nasal epithelial cells.Toluene diisocyanate (TDI) is a major cause of occupational asthma and rhinitis. Shoseiryuto (SST) is one of the traditional herbal medicines (Kampo medicine) and has long been used as a natural medicine for allergic diseases such as allergic rhinitis (AR) and asthma. Recent studies have shown that the expression and release of IL-33, which regulates the T H2 cytokine-dependent response [Allergic rhinitis (AR) is induced by allergens such as ragweed and other pollens, mites, and fungi. It is one of the most common allergic inflammatory diseases, affecting more than 600 million people worldwide ,2. The iresponse ,4. EarlyAirway epithelial cells provide an important barrier function in the superficial layer of the airways, preventing the entry of harmful substances from the environment. In addition to this barrier function, the airway epithelium also plays a role in controlling inflammatory conditions by producing and secreting cytokines and chemokines that regulate various inflammatory responses .H2 cytokine production in various immune cells, including TH2 and mast cells [Among the inflammatory cytokines involved in the late phase of AR, IL-33 has been highlighted as one of the key cytokines. IL-33 belongs to the IL-1 family and activates the same intracellular signaling pathway as IL-1 and IL-18, with ST2 as a receptor ,7. It haIL-33 protein is expressed in the nuclei of nasal epithelial cells in the normal condition and is rapidly released into the nasal fluid after exposure to allergens ,9. In huToluene diisocyanate (TDI) is a leading cause of occupational asthma (OA) and is also known to induce allergic rhinitis . RegardiH2 cytokines, including IL-4, 5, and histamine H1 receptor (H1R) [Shoseiryuto (SST), called Xiao-quinglong-tang in China and So-cheong-ryong-tang in Korea, is one of the traditional herbal medicines (Kampo medicine) that has long been used as a natural medicine for allergic diseases, such as AR and asthma. SST has been used to improve both acute symptoms, including sneezing and rhinorrhea, and chronic symptoms, including nasal obstruction, in AR patients . In relaor (H1R) .In the present study, we show an ameliorative effect of SST on TDI-induced AR symptoms, which has not yet been clarified, and examined the relationship between the effect of SST and the IL-33 release from nasal epithelial cells.Sprague-Dawley (SD) male rats at the age of 5 weeks (120–130 g) were purchased from Nippon Bio-Supp. Center . Three or 4 rats were housed in a cage in the conditioned room . The rats accessed food  and water ad libitum. All efforts were made to minimize animal suffering and to reduce the number of animals used. This study was conducted with the approval of the Committee of Animal Care and Welfare of Showa University and in accordance with the guidelines established with the Committee of Animal Care and Welfare of Showa University . Each experiment was repeated two times.Eighteen rats were randomly assigned to tree groups. Control: distilled water; Allergic rhinitis (AR): TDI; AR + SST: TDI + SST. The AR and AR + SST groups were sensitized with a modified protocol of Zheng et al.  by droppPinellia ternata, Breitenbach), 3.0 g of Asiasarum root , 3.0 g of Cinnamon bark , 3.0 g of Ephedra herb (Ephedra sinica Stapf), 3.0 g of Glycyrrhiza , 3.0 g of Schisandra fruit (Schisandra chinensis Baillon), 3.0 g of Paeony root , and 3.0 g of Processed ginger .SST Lot. 2140019010) was kindly gifted by Tsumura and Co.  and prepared using the same procedures as previously reported [0019010 wAfter the last TDI stimulation, each animal was observed for 10 min, and the number of sneezes and the time spent nose scratching were scored.g for 10 min, and the supernatant was collected, stored at −80 °C, and used for measurement.Nasal lavage fluid (NLF) was collected from rats anesthetized with intraperitoneal administration of pentobarbital sodium . The trachea was incised, a polyethylene catheter with a diameter of 1 mm was inserted into the nasopharyngeal cavity, and 1 mL of PBS flushed from the nostrils was collected. The NLF was centrifuged at 400× Subsequently, blood was collected from the inferior vena cava after laparotomy.IL-33 concentrations in NLF and serum were examined in triplicate using an ELISA test kit  according to the manufacturer’s protocol.g for 10 min. The serum was withdrawn, and the histamine content was measured using an ELISA kit  according to the manufacturer’s protocol.Blood was centrifuged at 400× After sampling NLF, the anesthetized rats were perfused intracardially with cold PBS (pH 7.4) and then perfused with 4% paraformaldehyde/PBS. The tissue, including the nasal cavity, was excised and cut into 20 μm thickness by a cryostat . Then immunostaining process was conducted: rinse with PBS 3 times, blocking (10% goat serum with 0.3% Triton X-100 in PBS) for 2 h at room temperature, incubation with primary antibody  for 48 h, washing with PBS 3 times, incubation with 2nd antibody . Nuclei were counter-stained with DAPI . Immunofluorescent images were obtained with a confocal microscope .g, 20 min). Then, TDI-HSA and control-HSA solutions were dialyzed with a dialysis tube  for 3 days with 4 L of 0.1 M ammonium carbonate. TDI-HSA and control-HSA were precipitated with an equal volume of 20% trichloroacetic acid and then redissolved with 1 M sodium hydroxide. The solution was dialyzed again . Synthesized TDI-BSA was visualized by Coomassie blue staining after native SDS-PAGE.TDI-HSA was prepared as reported previously . Briefly2 and 95% air at 37 °C. Twelve hours after stimulation with TDI-HSA, HNEpCs were fixed with 4% sucrose-containing 4% paraformaldehyde  for 20 min. The fixed cells were permeabilized with 0.2% Triton X-100/PBS  for 5 min and blocked with 10% goat serum/PBS for 1 h. Then, an anti-IL-33 antibody  was applied overnight at 4 °C. IL-33 was visualized by isotype-specific secondary antibody conjugated with Alexa 488 . A fluorescent microscope  was used to obtain fluorescent images.Human Nasal Epithelial Cells (HNEpCs)  were cultured on poly-L-lysine coated cover glasses  in Airway Epithelial Cell Growth Medium (Promocell) and maintained in 5% COp-value < 0.05 was considered statistically significant.All the values are expressed as means and standard error of the mean (SEM). Each data set was firstly analyzed using the Kolmogorov–Smirnov test to determine whether the data were normally distributed or not. One-way ANOVA followed by Tukey’s multiple comparisons or Kruskal-Wallis tests was used for normally distributed or non-normally distributed data, respectively, using EZR  . p-valueRats were sensitized to/provoked with TDI and administered SST in the schedule, as shown in Provocation with TDI after sensitization to the reagent clearly induced apparent allergic symptoms such as reddening and swelling in the nose of rats A. Rats tNext, the number of sneezes and duration of nose scratching for 10 min immediately after provocation were determined. In TDI-sensitized AR rats, the total number of sneezes was 12.3 ± 2.58 while 0.17 ± 0.17 in the control rats B. AdminiSerum histamine levels were also measured as a marker of inflammatory levels in rats. As expected, serum histamine levels were dramatically raised in TDI-sensitized rats, which was suppressed by SST treatment  D. These We next determined IL-33 levels in NLF and serum. TDI-sensitized AR rats showed significantly elevated IL-33 levels in serum and a trend toward elevation in NLF, while control rats had similar levels of IL-33 in both serum and NAL as control rats. A  was prepared. After 1 h of TDI-HSA treatment, the percentage of cells expressing IL-33 in the nucleus decreased A,B. PretThis study showed that SST, a Kampo medicine, suppressed symptoms associated with TDI-induced AR at least partially via inhibiting IL-33 release from the nuclei of nasal epithelial cells both in vivo and in vitro experiments.H2 cytokines such as IL-4/5, which are growth factors for eosinophils, basophils, and eosinophils. Recent studies have revealed an additional important step: the expression and release in epithelial cells of cytokines such as IL-33, which regulates the TH2 cytokine response [The inflammatory response of the nasal mucosa consists of the following phases: IgE-mediated immediate mast cell response. Late-phase reaction and persistent allergic inflammation through mobilization of T cells expressing Tresponse . Criticaresponse . HoweverH2-type allergic reactions, affecting the differentiation of naive CD4+ T cells to Th1 or TH2 cells. SST decreased the expression of IL-4 mRNA, which is essential for TH2 cell development, and increased IFN-γ expression, which is important for Th1 cell development in mice [Dermatophagoides farinae [TDI is a recognized irritant to the human body and is one of the leading causes of occupational allergic disease in industrialized countries . It has  in mice ,25. Tana farinae . We also farinae . One ele farinae . SST see farinae ,29. Naka farinae . In addiIL-33 is synthesized as the full-length form and localized in the nucleus in epithelial cells. The full-length IL-33 is cleaved to generate a high-activity form when cells are exposed to environmental aeroallergens. That process has an important function in the rapid and efficient induction of allergic type 2 responses . The steRecent basic research on herbal medicines has revealed many useful actions and their molecular mechanisms. Since IL-33 release from nasal epithelial cells is considered to be a common mechanism of AR exacerbation, the use of SST is highly promising not only for AR caused by TDI but also for AR caused by other allergens. A limitation of this study is that it is not yet clear how SST blocked IL-33 release from epithelial cells. Further studies are needed to clarify its molecular mechanism. It is likely that SST will be used more and more in clinical practice in the future because of the advantages of herbal medicine, which has fewer side effects and has a combined action.


This study aimed to verify whether curcumol combined with paclitaxel exerted synergistic antiproliferative and proapoptotic effects in MDA-MB-231 mammary cancer cells.The effects of different concentrations of CC, PTX, and their combination on the proliferation of MDA-MB-231 mammary cancer cells were determined by CCK-8 laboratory tests. Combination index (CI) was calculated using CompuSyn software. Colony formation assays, Hoechst 33258 immunofluorescence staining, and flow cytometry were carried out to observe proliferation and apoptosis in each group. The protein expression of PCNA, Bcl-2, Bax, ZBTB7A, p-p65, and NF-ƙB p65 was detected by western blotting. The xenograft tumor volume and body mass of nude mice were measured. Immunohistochemistry was used to detect the expression of PCNA , NF-B p65 and ZBTB7A. TUNEL and DAPI staining were used to detect the apoptosis of tumor cells.in vivo and in vitro. Expression of PCNA, Bcl-2, ZBTB7A, p-p65, and NF-B p65 was lowest in the CC+PTX group, while the expression of Bax was highest. The growth of xenograft tumors in the CC+PTX group was most notably suppressed. Immunohistochemistry showed that expression of PCNA, ZBTB7A, and NF-ƙB p65 was the lowest in the CC+PTX group.Curcumol combined with paclitaxel exerted a significant inhibitory effect on proliferation of MDA-MB-231 cells in the CCK-8 laboratory test. Hoechst 33258 immunofluorescence staining, flow cytometry, TUNEL, and DAPI apoptosis staining demonstrated that cell apoptosis was the highest in the CC+PTX group Curcumol combined with paclitaxel exerted a synergistic antiproliferative and proapoptotic effect on triple-negative breast cancer cells  The incidence and mortality of mammary cancer rank first among female malignancies worldwide . Triple-Curcumol (CC) is a natural sesquiterpenoid. Several studies have confirmed that CC exerts significant anti-inflammatory, antiviral, antioxidant, and antifibrotic effects . In termA prominent hallmark of malignant tumors is the unrestricted proliferation and inhibited apoptosis of tumor cells. PCNA, Bax, and Bcl-2 have been confirmed by a large number of studies to be important proliferation and apoptosis proteins that can reflect the anti-tumor effect of drugs in breast cancer. Studies have confirmed that the NF-κB signal transduction pathway is directly or indirectly involved in inflammatory responses, cell proliferation, apoptosis, angiogenesis, immunosuppression, therapy resistance, and metabolic processes , 10. HuaPrevious studies have confirmed that chemotherapy drugs combined with monomers of natural medicines can more effectively inhibit tumor cell proliferation, promote apoptosis, inhibit metastasis, and improve efficacy , 17. TheCell culture and reagents °C with 5% CO2. CC powder (purity≥98%) and PTX for injection were procured from the National Institute for the Control of Pharmaceutical and Biological Products  and Yangtze River Pharmaceutical Group , respectively. The mother liquor of CC was prepared by dissolving in absolute ethanol. The concentration of ethanol in the working solution subsequently administered to each treatment group was less than 0.1%. Antibodies against Bcl-2 (B-cell lymphoma-2), Bax (Bcl-2 associated X), PCNA, NF-κB p65, Phospho-NF-κB p65 (p-p65), and GAPDH were obtained from Cell Signaling Technology, Inc. . Antibodies against ZBTB7A and HRP-labeled goat anti-rabbit IgG were obtained from Abcam, Inc.  and Beyotime , respectively.MDA-MB-231 cells were purchased from the Cell Bank of the Chinese Academy of Sciences . The cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum , 1% penicillin, and streptomycin. The standard culture atmosphere in the humidified incubator was maintained at 37 Viability laboratory test and synergy analysis3 cells per well. Twenty-four hours later, the cells were treated with different concentrations of CC , PTX  and CC+PTX . 24 hr, 48 hr, and 72 hr later, the optical density of each well was examined by a microplate reader at 450 nm. GraphPad Prism 8.0 was used to determine the half-maximal inhibitory concentration (IC50) of CC and PTX on MDA-MB-231 cells. Compusyn software  was used to evaluate the joint efficacy of CC and PTX based on the Chou-Talalay methodology  laboratory tests. Cells in the logarithmic phase of growth were resuspended in medium and seeded into 96-well plates at 3×10hodology . The synColony formation assayA total of 250 MDA-MB-231 cells resuspended in the medium were seeded into each well of 6-well plates. Twenty-four hours later, the cells were treated according to their groups. After 6 hr, the cells were cultured in a medium without drugs for 2 weeks. After fixing and staining, the clones were counted under an optical microscope and photographed with a camera. Colony formation rate (%)=(number of colonies/250)×100%.Hoechst immunofluorescence staining laboratory testTo observe apoptosis and morphological changes, we submitted the cells of each group to Hoechst 33258  immunofluorescence staining. Forty-eight hours after drug treatment, the cells were fixed and stained according to the instructions of the kit, and then, the difference in apoptosis and the change in cell morphology between the groups were observed under a fluorescence microscope. Five fields were randomly selected from each group to observe the apoptotic rate (AR) by fluorescence microscopy. AR (%)=number of apoptotic cells/total number of cells×100%.Flow cytometry To assess the differences in the apoptotic rates among the four groups, we submitted the cells of each group to a specified drug treatment. After 48 hr, the collected cells were double-stained using 5 μl Annexin V-APC and 5 μl 7-AAD for 15 min in the dark. Then, the apoptotic rate was detected by flow cytometry . Apoptosis rate (%)=(the number of early apoptotic cells+the number of late apoptotic cells)/total number of cells×100%.Western blotting°C overnight) and HRP-labeled goat anti-rabbit IgG for 1 hr in sequence. Enhanced chemiluminescence  substrate and ImageJ software were utilized to determine the relative protein expression. The gray value of the GAPDH band was used as the internal reference, and the ratio of the gray value of the target protein to the internal reference was used as the relative expression level.After treatment with the indicated drugs for 48 hr, the proteins of each group were extracted in a radioimmunoprecipitation laboratory test  lysis buffer and phenylmethylsulfonyl fluoride . A BCA protein assay kit  was used to measure the protein concentration of each group. Protein (30 μg per well) was loaded into gels and separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, the separated proteins were transferred to polyvinylidene fluoride membranes  and blocked with a Western blocking solution  for 15 min. The membranes were then incubated with primary antibodies for 16 hr  according to the random number table method based on the tumor volume of the nude mice. The four groups of nude mice were intraperitoneally injected with the same volume of PBS, CC (100 mg/kg), PTX (10 mg/kg), or the combination of CC+PTX. The procedures were repeated every 2 days. The body mass of the nude mice and the length and width of the xenograft tumors were measured and recorded every 4 days. All nude mice were sacrificed 24 days after subcutaneous transplantation, and then, the weight of the xenograft tumors was precisely measured after careful dissection. Tumor volume was evaluated as follows: Volume (cm3)=width2 (cm2)×length (cm)/2.Twenty-eight 5-week-old female nude mice (14.8~20.9 g) were procured from the Experimental Animal Center of Guangxi Medical University. All of the animal experiments were conducted strictly in accordance with the related principles of the Ethics Committee of Guangxi Medical University Cancer Hospital. One hundred microliters of PBS containing 6×10Immunohistochemistry 2O2. Then the samples were incubated with primary rabbit PCNA, rabbit ZBTB7A, and rabbit NF-κB p65 antibodies overnight at 4 °C. The next day, all sections were incubated with horseradish peroxidase-conjugated secondary antibodies . Finally, a color reaction was carried out using 3,3’-diaminobenzidine . Normal rabbit serum was used as a negative control instead of the primary antibody. The tan or tan granules located in the nucleus are positive cells. Five different fields of view were randomly selected in the slice, and 200 breast cancer cells were selected for counting in each field of view, for a total of 1000 cells, and the proportion of positive cells was calculated. The xenograft tumor tissues were paraffin-embedded 24 hr later, and then, 4-μm-thick tissue sections were generated. All sections were dewaxed with xylene and dehydrated with different concentrations of ethanol. Subsequently, tissue sections were blocked with goat serum after thermal antigen retrieval and blocking of endogenous peroxidase with 3% HTUNEL and DAPI apoptosis staining Tissue sections of the nude mice were heated, dewaxed, and dehydrated. Apoptosis in each group was observed strictly according to the instructions of the Tunel (TdT-mediated dUTP nick end labeling) Apoptosis Staining Kit . Next, the nuclei were repeatedly stained with 4’,6-diamidino-2-phenylindole (DAPI). Five random areas under high-power fields of view were selected from each slice to observe the apoptotic rate (AR) by immunofluorescence microscopy. AR (%)=number of apoptotic cells/total number of cells×100%.Statistical analysis in vitro experiments were repeated at least three times. The measurement data for all the experimental data are presented as x̄±s. One-way analysis of variance was applied to compare differences between groups. SPSS 21.0 was applied to statistically analyze all experimental data. P<0.05 was defined as a statistically significant difference.All CC and PTX decreased the viability of MDA-MB-231 cells 50 values of CC and PTX were 558.6 μM and 4.4 μM, respectively.A CCK-8 proliferation laboratory test was used to elucidate the effects of CC and PTX on viability of MDA-MB-231 cells. The results demonstrated that CC and PTX decreased the viability of MDA-MB-231 cells in a dose- and time-dependent manner . After MCC combined with PTX generated a synergistic effect on decreasing the viability of MDA-MB-231 cells 50 of each drug and treated the cells for 48 hr. CC combined with PTX also exerted the same effect on decreasing the viability of MDA-MB-231 cells  in CC group and PTX group was obviously suppressed, whereas the CC+PTX group showed the most significant suppression . The underlying mechanism may be related to down-regulation of ZBTB7A expression via the NF-κB signaling pathway.AM and CW designed the entire experimental route. CW and QQ guided implementation. AM conducted the experiment, analyzed data, and drafted the manuscript. WY provided literature search assistance. CW and QQ provided fund support.The authors report no conflicts of interest in this work.


Salmonella Typhimurium is an invasive enteric pathogen that causes gastroenteritis in humans and life-threatening systemic infections in mice. During infection of the intestine, S. Typhimurium can exploit nitrate as an electron acceptor to enhance its growth. However, the roles of nitrate on S. Typhimurium systemic infection are unknown. In this study, nitrate levels were found to be significantly increased in the liver and spleen of mice systemically infected by S. Typhimurium. Mutations in genes encoding nitrate transmembrane transporter (narK) or nitrate-producing flavohemoprotein (hmpA) decreased the replication of S. Typhimurium in macrophages and reduced systemic infection in vivo, suggesting that nitrate utilization promotes S. Typhimurium systemic virulence. Moreover, nitrate utilization contributes to the acidification of the S. Typhimurium cytoplasm, which can sustain the virulence of S. Typhimurium by increasing the transcription of virulence genes encoding on Salmonella pathogenicity island 2 (SPI-2). Furthermore, the growth advantage of S. Typhimurium conferred by nitrate utilization occurred only under low-oxygen conditions, and the nitrate utilization was activated by both the global regulator Fnr and the nitrate-sensing two-component system NarX-NarL. Collectively, this study revealed a novel mechanism adopted by Salmonella to interact with its host and increase its virulence. Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative intracellular pathogen that typically causes mild self-limiting gastroenteritis in humans; however, mouse infection always results in life-threatening systemic infection with disease symptoms that resemble typhoid fever in humans  ethylenediamine). Finally, the total concentrations of nitrate and nitrite were calculated by measuring the absorbance of the deep purple azo chromophore at 540 nm  at excitation wavelengths of 488 (F488) and 440 nm (F440). Intracellular pH was calculated from the ratios of emission fluorescence intensities upon excitation at 488 and 440 nm (F488/F440) using the pHin  standard curve that was prepared using the following method. First, 500 mL of N-minimal medium was aliquoted into five 100 mL portions, and the pH values were adjusted to 4, 5, 6, 7, and 8. The bacterial culture was centrifuged and collected, after which it was suspended in the same volume of N-minimal mediums with different pH, and the OD600 of the bacteria was adjusted to 0.3. Valinomycin (1 μM) and nigericin (1 μM) were then added to the bacterial suspensions. The bacterial suspensions were gently vortexed to achieve a balance between pHin and extracellular pH. The bacteria were collected by centrifugation and suspended in the same N-minimal medium with different pH values. A 20 μM concentration of BCECF-AM was added to the bacterial suspensions at 30 °C for 20 min. The bacteria were collected by centrifugation, washed three times by using the N-minimal medium with the same pH value, and suspended again. The fluorescence intensity of the bacterial suspension was measured at an emission wavelength of 535 nm with excitation wavelengths of 488 nm and 440 nm. The fluorescence intensity (F) is the ratio of F488 to F440, and the standard curve was plotted with pH values as the x-axis value and lgF as the y-axis value [is value ,17.2SO4, 1 mM KH2PO4, 5 mM KCl, 0.5 mM K2SO4, 10 μM MgCl2, 10 mM Tris-HCl (pH 7.5), 0.5% (v/v) glycerol, and 0.1% (w/v) casamino acids). To obtain the bacteria in stationary phase, the overnight-cultured bacteria were sub-cultured in fresh N-minimal medium  at a 1:100 inoculation ratio for 6 h. S. Typhimurium cells were collected by centrifugation at 10,000 rpm for 3 min and immediately frozen in liquid nitrogen. Total RNA was extracted using TRIzol reagent  according to the manufacturer’s instructions. Residual DNA in the total RNA samples was removed using a RNeasy Mini Kit . The final RNA concentration was determined by using a NanoDrop 2000 spectrophotometer .For qRT-PCR analysis, bacteria were grown overnight in N-minimal medium . The primers used for qRT-PCR analysis are listed in S. Typhimurium that resides in the mouse tissues, around 0.2 g of the S. Typhimurium-infected tissues were quickly harvested and homogenized by mixing with 0.5 mL of DNase/RNase-free water. The total RNA from the tissues and bacteria were extracted by using the TransZol Up Plus RNA Kit , which provided an effective solution that can simultaneously extract the RNA from both the animal tissues and bacteria. When performing qRT-PCR, a maximal cDNA amount should be used because the bacterial RNA takes up a small proportion of total RNA.To quantify the gene expression of Escherichia coli BL21 (DE3) cells. The DE3 bacteria harboring the pET28a-gene plasmid were cultured overnight in LB medium and sub-cultured in fresh LB medium at an inoculation rate of 1:100. When the OD600 was close to 0.6, 1 mM isopropylthio-β-galactoside (IPTG) was added to the LB medium to induce the expression of the 6 × His-tag protein. Sodium nitrate (30 mM) was added to the LB medium when expressing the NarL-6 × His protein to ensure that the protein was phosphorylated in response to nitrate. The protein was purified from the lysate supernatant from DE3 cells using a His-Tagged Protein Purification Kit . Protein concentration was determined according to the Bradford method, and the purified protein was stored at −80 °C.The constructed pET28a-gene plasmid was transformed into EMSA was performed as previously described with some modifications. Briefly, 400 bp of DNA fragments consisting of the 300 bp upstream sequence and the first 100 bp of the target genes were amplified by PCR with high-fidelity enzymes and purified from gels, as well as the DNA fragments of 16S rDNA (negative control). The promoter fragments (50 ng) and different concentrations (0–1.6 μM) of the purified protein were incubated at room temperature for 30 min in a 20 μL solution containing EMSA binding buffer (20 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, 1 mM dithiothreitol, and 5% glycerol). Samples were then loaded onto a 6% polyacrylamide gel immersed in 0.5 × Tris-Borate-EDTA for electrophoresis. DNA fragments were stained with GelRed .t-test, log-rank (Mantel–Cox) test, or Mann–Whitney U test was used to analyze significant differences between the two groups according to the test requirements. Differences were considered significant at p < 0.05 .Data were analyzed using GraphPad Prism . The data presented in each figure or table were obtained from three independent biological experiments (in vitro experiments) or a combination of two independent biological experiments (in vivo experiments). The Student’s S. Typhimurium-infected macrophages and the liver and spleen of S. Typhimurium-infected mice are significantly increased, and nitrate utilization contributes to the intracellular replication and systemic infection of S. Typhimurium. The utilization of nitrate can increase the acidification of S. Typhimurium cytoplasm, which is a crucial signal to activate the expression of SPI-2 virulence genes. Nitrate utilization of S. Typhimurium is activated by both Fnr and NarX-NarL (see S. Typhimurium during systemic infection). These findings reveal a new mechanism related to the acidification of the S. Typhimurium cytoplasm and also provide new insights into the course of Salmonella systemic infection and the interactions between Salmonella and its host.This work reports that nitrate levels in S. Typhimurium cytoplasm needs to be addressed in the future study, and whether the metabolic products of nitrate, such as nitrite or ammonium, contribute to the virulence of S. Typhimurium during systemic infection is worth exploring. Salmonella. enterica serovar Typhi (S. Typhi), the human-limited serovar, can cause lethal typhoid fever in humans via replicating inside human macrophages. The effect of host-derived nitrate on the virulence of S. Typhi can be assessed in the human macrophages and humanized mice if possible.How the nitrate utilization acidifies the


Metformin, the first-line oral antidiabetic medicine, has shown great antineoplastic potential in various cancer types, despite an unclear mechanism. This study aimed to elucidate the possible mechanism of metformin as a chemotherapy agent with less reproductive and genetic toxicity in human endometrial cancer. The type I endometrial carcinoma cell lines Ishikawa and RL95-2 were treated with metformin. Cell functions, such as proliferation, migration, and invasion, were analyzed. Flow cytometry was performed for cell cycle and apoptosis analyses. Simultaneously, RT-qPCR and western blotting were performed to explore the possible mechanism. Moreover, YAP1 knockout Ishikawa cells were established via lentivirus to demonstrate the underlying mechanism. The results showed that metformin mediated Ishikawa and RL95-2 cell growth inhibition in a dose- and time-dependent manner. The IC50 values of metformin in Ishikawa and RL95-2 cells were 10 mM and 8 mM, respectively. The migration and invasion abilities were also inhibited in the metformin-treated group using wound healing assays and transwell migration and invasion assays, and Ishikawa and RL95-2 cells were arrested in the G1 or G2 phase, respectively. Moreover, the cell proportions of cells in both early and late apoptosis stages were dramatically elevated when treated with metformin, as was the ratio of Bax/Bcl-2 expression. Additionally, the expression levels of YAP1 mRNA and protein in the treatment group were much lower than those in the control group. The cellular behaviors of YAP1 knockout Ishikawa cells were similar to those in the metformin-treated group. Our results demonstrated that it is an attractive alternative to cytotoxic chemotherapy in human endometrial cancer, and YAP of the Hippo pathway may be a potential molecular target. This study provides novel ideas for the adjuvant therapy of endometrial cancer patients, especially for women with strong fertility desires and demands. Endometrial cancer is the most frequently occurring gynecologic malignancy in Western countries. It has been reported that since 2008, the incidence of endometrial cancer has increased by 21% in the United States, as well as doubling the death rate over the past two decades , chemothThe Hippo signaling pathway is an evolutionarily conserved signaling transduction and regulator involved in cell differentiation, tissue regeneration, organ development, and immune modulation . The corMetformin, the most widely used antidiabetic drug , has beein vitro and the role of the Hippo signaling pathway in the process.The present study aims to explore the antineoplastic effect of metformin on endometrial cancer v/v) fetal bovine serum , 100 U/mL penicillin , and 100 μg/mL streptomycin  at 37°C in a humidified atmosphere with 5% CO2 in an incubator . Metformin  was dissolved in ddH2O and then diluted to a suitable concentration. YAP1-knockout (YAP1-KO) lentivirus (LV-YAP1-KO) based on the CRISPR/Cas9  system was purchased from Genechem, China. Ishikawa cells were transfected with LV-YAP-KO using HitransG P . Following selection with 5 μg/ml puromycin  for 72 h, single-cell clones were sorted, expanded, and validated as YAP1-KO Ishikawa cells by western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis.Human type I endometrial carcinoma Ishikawa and RL95-2 cells were previously donated by the Cancer Biology Research Center of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Cells were cultured in Dulbecco's modified Eagle medium (DMEM)-high glucose  containing 10% .Cell function assays included cell viability assays, flow cytometry analysis, wound healing assays, transwell migration assays, and The cell cycle was analyzed using flow cytometry. Cell pellets were resuspended in precooled 90% ethanol and then incubated at −20°C overnight. After washing and incubating in a water bath, the cells were stained with propidium iodide  for 20 min in the dark before flow cytometry analysis. Dual staining was performed for the apoptosis assay using an Annexin V-FITC/PI kit . Cells stained with FITC-conjugated Annexin V and PI were measured by flow cytometry.Well-spread cells in 24-well plates were scratched by a pipette tip after FBS starvation for 12 h and then observed every 24 h using an inverted microscope . The relative wound areas were quantified and calculated by ImageJ .μL FBS-free medium were seeded in the top chambers of transwells  and Matrigel-coated transwells , and the medium on the bottom was supplemented with 20% FBS. After 48 h of culture, cells in the chambers were fixed with 4% paraformaldehyde, stained with crystal violet , and then counted under a 20× microscope .For the transwell migration and invasion assays, 10,000/mL cells in 100 β-actin (66099-1-Ig) were purchased from Proteintech, China. Anti-YAP1 (#14074) and anti-phospho-YAP1 (#13008) were purchased from CST, United States. The secondary antibodies (1 : 2000) were purchased from Servicebio, China. The antibodies mentioned above were diluted at a ratio of 1 : 1000 unless stated otherwise.The concentrations of total protein extracted from the cells were quantified using a BCA assay . After separation using 10% SDS-PAGE, the proteins were transferred to PVDF membranes  and then incubated with the corresponding primary antibody and horseradish peroxidase-conjugated secondary antibody according to the manufacturer's instructions. Protein visualization was performed using an ECL kit . Protein expression was quantified using ImageJ software . Anti-LAST1 (17049-1-AP), anti-Bcl-2 (12789-1-AP), anti-Bax , anti-GAPDH (10494-1-AP), and anti-Premix Ex Taq  following the manufacturer's instructions. The mRNA expression was calculated using the 2−ΔΔCt method and normalized to GAPDH. The sequences of the primers were as follows: YAP1, 5′-ACTGGCTACGCAGGGCTA-3′ (F), 5′-TTTGAGTCCCACCATCCTGCTCCAG-3′ (R), GAPDH: 5′-TGAGTCCTTCCACGATACCAA-3′ (F), 5′-AGTCTTCTGGGTGGCAGTGA-3′ (R).Total RNA was isolated from the cells using NucleoZol reagent . Then, 500 ng of total RNA was used to synthesize cDNA using a reverse transcription kit , and qPCR was performed using TB Green t-test was used for comparisons between two groups, and analysis of variance (ANOVA) was used for comparisons among three groups. Two-tailed hypothesis tests were performed. P < 0.05 was considered significant. Each experiment was performed in triplicate and repeated at least three times independently.Statistical analyses were performed by using SPSS version 26.0 , and data visualization was carried out using GraphPad Prism 8.0 . All data are presented as the mean ± standard deviation (SD). Student's independent Ishikawa and RL95-2 cells were incubated with metformin at different concentrations , and the cell proliferation ability was detected using CCK-8 assays. As shown in Cell apoptosis rates were examined in Ishikawa and RL95-2 cells after 48 h metformin treatment  by Annexin V-FITC/PI flow cytometry. FITC + PI- cells were regarded to be in the early stage of apoptosis, and FITC + PI + cells were in the late stage of apoptosis. As shown in Figures P < 0.01), demonstrating that metformin inhibited Ishikawa in vitro migration in a dose-dependent pattern , the number of penetrating cells was much lower than that in the 10 mM metformin group (55.7 ± 33.3) and the 20 mM metformin group (20.6 ± 14.0) (P < 0.01)  to investigate cell migration and invasion. Since confluence growth was absent in RL95-2 cells, the wound healing assay was only performed in Ishikawa cells. The relative areas between the scratches in the control group were positively correlated with exposure time, and the wound was almost healed after 48 h of treatment. In the metformin treatment group, the speed of wound healing was significantly slower, especially with a higher concentration of metformin, which indicated an inhibitory effect of metformin on Ishikawa cell migration Figures . The res Figures . A more  Figures . Then, t Figures . Similar Figures .The Hippo signaling pathway may be an antitumor target of metformin in endometrial carcinoma cells.in vitro invasion in Ishikawa cells, and the results demonstrated the obvious inhibition of invasion in YAP1-KO Ishikawa cells  for 48 h to explore the possible mechanism of the antitumor effect of metformin. The mRNA and protein expression of YAP1 and LATS in Ishikawa cells were detected using qRT-PCR and western blotting. The relative protein level of p-YAP1/YAP1 increased obviously in the metformin-treated group Figures . Additio Figures . Equally Figures .In the current study, we found that metformin was able to inhibit cell proliferation, migration, and invasion, as well as induce cell cycle arrest and apoptosis in human endometrial cancer cells. Moreover, YAP, the core of the Hippo signaling pathway, might be a potential target for the antineoplastic therapy with metformin in endometrial cancer .in vitro.First, Ishikawa and RL95-2 cells were treated with various concentrations of metformin at different times and analyzed by CCK-8 assay to seek a suitable treatment concentration and time. Cell cycle arrest in G1 and G2 phases can be observed by flow cytometry, and metformin is likely to interfere with the process of DNA replication. The results of Annexin V/PI dual staining flow cytometry also proved that metformin induced both early and late stages of apoptosis, which was further reinforced by western blotting. Moreover, the decrease in Bcl-2/Bax in the metformin-treated group suggested the possible participation of the mitochondrial pathway. In addition, more cell functions were investigated through wound healing tests and transwell analysis, and significant inhibition of cell migration and invasion was observed. The above results indicated the significant suppression effects of metformin on endometrial cancer cells Metformin has shown great potential for antidiabetic, antiphlogistic, antilipemic, and antineoplastic properties. It has been reported that metformin reduces the incidence and death rate and improves the prognosis of multiple malignancies, including hepatocellular , pancreaThe Ishikawa and RL95-2 cell lines both originated from type I endometrial cancers, which are hormone-dependent and caused by cumulated estrogen with the lack of the antagonism of progesterone. Type I endometrial cancer is usually well-differentiated adenocarcinoma, normally accompanied by obesity, diabetes, and infertility, and secondary to atypical hyperplasia of the endometrium . Interesin vitro studies, with an IC50 of 8 to 10 mM, were much higher than those in animals, which were between 40 and 70 μM of the plasma concentration in veins after the administration of a therapeutic dose [Drug safety is always the focal concern of health care providers. Metformin has been proposed to improve the clinical outcomes of fertility in females suffering from polycystic ovary syndrome (PCOS) with insulin resistance . Comparetic dose . A similtic dose , but it in vitro cell experiments, and more animal studies are needed to reinforce our current results and conclusions. Moreover, the functional routes of metformin in endometrial cancer are complicated and cross-linked, and the Hippo signaling pathway may not be unique. The exact signaling pathway network remains to be explored. The lack of comparison of metformin and traditional antitumor drugs such as paclitaxel can be another limitation. Whether metformin can achieve a similar antitumor effect as other drugs is still unclear. More mechanistic studies with a better design are needed to investigate the anticancer effect of metformin on endometrial cancer in the future.There were still limitations in this study. We only detected the expression of a few proteins and mRNAs involved in apoptosis and the Hippo pathway, and the exploration of the mechanism was not comprehensive and deep enough. In addition, our conclusions were based on In conclusion, metformin treatment in human type I endometrial carcinoma Ishikawa cells and RL95-2 cells can inhibit proliferation, migration, and invasion, block the cell cycle, and promote apoptosis, demonstrating that metformin is an attractive alternative to cytotoxic chemotherapy in human endometrial cancer and that YAP of the Hippo pathway may be a potential molecular target. This study provides novel ideas for the adjuvant therapy of endometrial cancer patients, especially for women with strong fertility desires and demands.


Chronic viral hepatitis is a major global public health problem. The guidelines suggest the long-term performance of regular ongoing liver examinations to monitor liver inflammation and screen for hepatocellular carcinoma. However, the effects of regular liver examinations on health-related quality of life (HRQoL) have not been adequately evaluated. Therefore, this study evaluated the effects of regular ongoing examinations on the quality of life of patients with hepatitis.A cross-sectional study was conducted from October to December 2016 in four hospitals in northern Taiwan. A hepatitis pay-for-performance (P4P) program was launched in 2010 to ensure that hepatitis patients have regular ongoing liver examinations. The study group consisted of patients who joined and stayed in the program for more than one year. The study assessed HRQoL utilizing the five-level version of the EuroQol-5 Dimension (EQ-5D-5L) and the EuroQoL visual analog scale (EQ-VAS). The responses for the EQ-5D-5L in hepatitis patients were transformed into the EQ-5D index according to the Taiwanese population’s value set. Sociodemographic and clinical characteristics were collected by questionnaire, and descriptive statistics were presented. A two-part model and generalized linear model with a Poisson distribution and a log link function, respectively, were used to examine the associations of the EQ-5D index and EQ-VAS score with participation in the hepatitis P4P program. We applied propensity score weighting with inverse probability weighting to control for selection bias.P < 0.001). However, the differences in the EQ-5D index scores between the study and control groups were not significant.In all, 508 patients  were enrolled in this study. The mean  reported EQ-5D index and EQ-VAS scores were 0.93 (0.12) and 75.1 (13.8), and the median  values were 1 (0.108) and 80 (15), respectively. The study group had a moderately significantly higher EQ-VAS score (mean ratio = 1.029, Patients with hepatitis partially benefited from receiving hepatitis P4P in Taiwan, which featured regular ongoing liver examinations, in that their EQ-VAS scores were enhanced but not their EQ-5D index scores. Viral hepatitis is a major global public health problem, and mortality from viral hepatitis has increased by 22% from 2000 to 2015 globally. Hepatitis B (HBV) and hepatitis C (HCV) are the major forms of viral hepatitis and are responsible for 96% of hepatitis-related mortality. In 2015, the World Health Organization (WHO) estimated that 257 million people 3.5%) were living with chronic HBV infection and 71 million people (1%) were living with chronic HCV infection .5% were , and HCVTaiwan is an endemic area for HBV infection with a prevalence of approximately 15–20% before the massive vaccination program launched in June 1984 . A recenThe P4P program is a reimbursement model, different from fee-for-service, that provides extra financial rewards to physicians and medical institutes based on their performance on quality measures. The hepatitis P4P program in Taiwan aims to improve the quality of patient care by increasing the implementation of guidelines that suggest preventive services and regular liver examinations twice a year .Some studies have presented the effect of the hepatitis P4P program as related to the patient’s utilization rate of services and clinical outcomes. The results indicate that the P4P program improves adherence to a guideline-based examination schedule; however, the change reported is minimal . FurtherThe WHO defines QoL as an individual's perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns. Those aspects of self-perceived well-being that are related to or affected by the presence of disease or treatment are defined as health-related quality of life (HRQoL) . The US Given that the primary activities rewarded by the hepatitis P4P program in Taiwan are regular examinations, the incentives impact continuous follow-up by physicians . In pracThe National Health Insurance Administration (NHIA) is a mandatory single-payer health insurance program in Taiwan that provides comprehensive medical care to greater than ninety-nine percent of Taiwanese residents. The NHIA provides insurance-covered liver examinations to all patients with chronic HBV and HCV infection every 6 months. However, the attendant rate of such preventive services was only 22% in 2009 . In 2010This program is open to physicians specializing in gastroenterology, family medicine, or pediatrics who provide treatment for patients infected with HBV or HCV. Once they have received continuous follow-up at the same medical facility twice with a 6-month interval, patients can be enrolled in P4P by their physicians. The follow-up must include serum GOT/GPT tests and abdominal sonography. Patients participate in the program at their will. On enrollment, patients receive health education regarding viral hepatitis via the physician or disease manager. As they enroll patients, physicians become eligible for four types of extra payments per capita: a new enrollment payment (U.S.$3); a payment for subsequent follow-up every 6 months (U.S.$3); and a payment for (1) referral of a patient with a newly screen-detected hepatic tumor, for whom the tumor was diagnosed as an early-stage (The American Joint Committee on Cancer stage I or II) HCC by subsequent examinations (U.S.$17), and (2) confirmation of the referred hepatic tumor as an early-stage HCC (U.S.$17) or detecting and diagnosing a hepatic tumor as an early-stage HCC (U.S.$34). For care continuity, the patient must be registered through the NHIA website when joining the P4P and tracked and followed by the same physician or medical institute. Any patient referral must involve an online transfer procedure via the NHIA website. A patient who is lost to follow-up by his assigned physician or medical institute for more than one year is dropped from the program. If the follow-up rate among enrolled patients falls below 20%, the NHIA will cut the physician's payment the subsequent year. The enrollment rate was approximately 38% in 2016 . Low enrThis cross-sectional study was performed from October to December 2016, during which we collected data from patient surveys and medical record reviews at the gastroenterology clinics of four hospitals in northern Taiwan. Inclusion criteria were as follows: (1) patients infected with chronic hepatitis B and/or hepatitis C, and (2) patients aged 18 years or older. A previous study revealed that the duration of enrollment in the P4P program was associated with patient outcomes , indicatWe collected patient sociodemographic characteristics, including age, sex, income , education attainment , and marriage status . The following clinical characteristics were recorded: nonhepatitis-related comorbidities diagnosed by a doctor, hepatitis complications , admission or emergency department visits related to viral hepatitis within the previous year, and type of viral hepatitis . The list of comorbidities in our survey questionnaire was obtained from the Elixhauser Comorbidity Index .Regarding the outcome measures, we chose to emphasize HRQoL rather than QoL because HRQoL focuses on overall quality of life with respect to physical or mental health and can be easily used to assess the patient’s perception of illness status and treatment outcomes . We adopThe EQ-5D essentially consists of two parts: the EQ-5D index and the EuroQoL visual analog scale (EQ-VAS). The EQ-5D index is a self-reported but objective, multidimensional health status evaluated in five parts, including mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Each dimension has five levels that roughly correspond to no, slight, moderate, severe, and extreme problems . In contWhen they came in for routine follow-up for chronic viral hepatitis, the study subjects were screened and selected by trained interviewers, who briefly introduced the study, obtained informed consent, and distributed self-writing questionnaires. If the respondents could not read or appeared unable to understand the questions, the interviewers were asked to read the entire questionnaire to the interviewees to ensure their comprehension. The EQ-5D was generally completed by patients in the P4P group after finishing at least the two times of regular examinations, because they were required to have participated in the program consecutively for more than one year. This research was approved by the ethics committee of the Institutional Review Board (IRB), Taipei Hospital, Ministry of Health and Welfare (IRB number IRC-IRB-4-0014-01-02).Descriptive statistics were used to evaluate the sociodemographic and clinical characteristics investigated. Subjects with missing values for the aforementioned variables were excluded. The variance inflation factor (VIF) was used to address the problem of multicollinearity between independent variables. A VIF above 10 indicated a high correlation, and the correspondent variable was removed . MultivaTo avoid selection bias regarding P4P program participation, we applied a propensity score weight (PSW) with an inverse probability weighting to weight patients in the P4P intervention and control groups . CompareP < 0.05 in all analyses.All analyses utilized SAS software version 9.4  except for the two-part model, which was analyzed by STATA 15. The results were considered statistically significant for We interviewed and collected data from 516 patients. Eight cases had missing data, seven involving income level and one involving incomplete EQ-5D scores, reducing the sample size of our study to 508. Table Table P = 0.01). After performing a subgroup analysis according to the clinical variables, the differences were also significant among the patients with comorbidities, patients with hepatitis-related complications, patients without admission within 1 year (for hepatitis-related events), patients with or without emergency department visits within one year and patients without admissions within one year (for hepatitis-related events), and other hepatitis (hepatitis C and hepatitis B/C coinfection). However, hepatitis P4P did not substantially change patients' EQ-5D index, regardless of subgroup.The Kolmogorov–Smirnov test revealed skewed distributions of the EQ5D utility scores and VAS scores. Accordingly, mean  and median  scores are presented. The mean (SD) EQ-5D index and EQ-VAS scores were 0.93 (0.12) and 75.1 (13.8), and the median (IQR) values were 1 (0.108) and 80 (15), respectively (data not shown). Table P = 0.05). EQ-VAS scores of the patients with income levels ranging from US$2,001 to $3,333 per month  and greater than US$3,333 per month  were higher than those of patients with income levels less than US$1,000. Married patients exhibited higher EQ-VAS score than unmarried patients  and patients with other marital statuses, including separated and divorced patients . Association between P4P participation and the EQ-5D index was nonsignificant in the regression model with PSW adjustment in addition to the other covariables. The EQ-5D index values are significantly associated with marital status, comorbidity, and type of hepatitis patient infected. Regarding marital status, widowed patients  and patients with other marital statuses  had lower EQ-5D index values than married patients. Patients who had any one of the comorbidities had lower index scores than those without . Patients infected with hepatitis B  exhibited higher EQ-5D index scores than patients infected with hepatitis C and coinfected with hepatitis B/C. The R squared values of the regression models for the EQ-5D index and EQ-VAS scores were 0.14 and 0.10, respectively.Table P < 0.001). The monthly income, education level, and marital status of the hepatitis patients also were significantly associated with the EQ-VAS score. Regarding clinical considerations, patients who had visited the emergency department within one year for hepatitis-related events showed lower EQ-VAS scores . P4P participation was not significantly associated with the EQ-5D disutility score in the two-part model after applying PSW adjustment and adjusting for the other covariables . The regression results revealed that the disutility score was associated with patients’ education level . The other factors did not show a significant effect in the analysis.Table To the best of our knowledge, this is the first study to investigate the effect of hepatitis P4P on HRQoL. Our results demonstrate that hepatitis P4P was associated with significantly higher EQ-VAS scores in chronic viral hepatitis patients. However, such interventions were not significantly associated with improvements in the EQ-5D index values.P = 0.05). We do not clearly understand whether hepatitis P4P participation achieves a minimal clinically important difference (MCID) or responsiveness if 2.25 is considered significant. Other studies that are not related to hepatitis care have shown an MCID for EQ-VAS scores ranging from 0.39 to 10.82 [Previous HRQoL studies that employed the EQ-5D have shown that the mean EQ-5D index scores vary from 0.37 to 0.93, and the range of the mean EQ-VAS scores has been reported to be between 57 and 88 , 52–58. to 10.82 , 60, 61.to 10.82 , 63. We The association between hepatitis P4P and EQ-VAS health scores may be due to regular and ongoing examinations, continuity of care, physician trust, and control of comorbidity. With regard to the first, hepatitis P4P in Taiwan is characterized by regular and ongoing essential examinations, as studies have shown that examinations can promote patients’ overall health. For example, the United Kingdom (U.K.) P4P programs that target general practitioners include measures related to examinations in different fields, and studies have shown that in the U.K., P4P affects patient outcomes  but not The following considerations may help explain the ineffectiveness of hepatitis P4P on the EQ-5D index. Previous studies have shown that hepatitis P4P, which purely targets increased regular examinations, does not sufficiently reduce incidences of admission or cirrhosis , which mIt is important to note the limitations of this study. First, the cross-sectional study does not permit analysis of the long-term relationship between P4P and QoL. Future studies should examine the abovementioned effect in a longitudinal manner. Second, regarding the validity and reliability of the EQ-5D-5L values obtained using Taiwanese value sets, few hepatitis patient studies exist in this regard because the Taiwanese value set has only been proposed in recent years . In one Patients with hepatitis partially benefited from receiving hepatitis P4P in Taiwan, featuring regular ongoing examinations, as evidenced by enhancement of their EQ-VAS scores but not their EQ-5D indexes.


SPRINTER assessed the efficacy and safety of SNG001 in adults hospitalised due to COVID-19 who required oxygen via nasal prongs or mask.Despite the availability of vaccines and therapies, patients are being hospitalised with coronavirus disease 2019 (COVID-19). Interferon (IFN)-β is a naturally occurring protein that stimulates host immune responses against most viruses, including severe acute respiratory syndrome coronavirus 2. SNG001 is a recombinant IFN-β1a formulation delivered to the lungs versus placebo, in terms of times to hospital discharge and recovery to no limitation of activity. Key secondary end-points were progression to severe disease or death, progression to intubation or death and death.Patients were randomised double-blind to SNG001 (n=309) or placebo (n=314) once daily for 14 days plus standard of care (SoC). The primary objective was to evaluate recovery after administration of SNG001 Median time to hospital discharge was 7.0 and 8.0 days with SNG001 and placebo, respectively (hazard ratio (HR) 1.06 (95% CI 0.89–1.27); p=0.51); time to recovery was 25.0 days in both groups (HR 1.02 (95% CI 0.81–1.28); p=0.89). There were no significant SNG001–placebo differences for the key secondary end-points, with a 25.7% relative risk reduction in progression to severe disease or death ; p=0.161). Serious adverse events were reported by 12.6% and 18.2% patients with SNG001 and placebo, respectively.Although the primary objective of the study was not met, SNG001 had a favourable safety profile, and the key secondary end-points analysis suggested that SNG001 may have prevented progression to severe disease. Although the primary objective was not met, potentially due to improved standard of care, SNG001 had a favourable safety profile and key secondary end-points analysis suggested that SNG001 may have impacted progression of COVID-19 to severe diseasehttps://bit.ly/3huTDX9 The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has highlighted the impact of respiratory viruses on mortality and morbidity, and their resulting pressures on healthcare provision. Despite the availability of vaccines and therapies, patients continue to be hospitalised with, and die from, coronavirus disease 2019 (COVID-19) [e.g. due to genetics, ageing, comorbidities or autoantibodies against type I IFNs , are at greater risk of severe viral lung disease [Interferon (IFN)-β is a naturally occurring protein that stimulates immune responses critical for the development of host protection against most viruses, including SARS-CoV-2 –6. It is disease –15. Impo disease . Overall disease .via nebuliser is to achieve a high local concentration within the lower respiratory tract, the site of SARS-CoV-2 infection [in vitro antiviral activity against SARS-CoV-2, including a number of variants of concern [Injectable and subcutaneous formulations of IFN-β have not demonstrated clinically meaningful effects in COVID-19 , possiblnfection . Inhalednfection  and to h concern .via nasal prongs or mask, but who did not need high-flow oxygen or ventilatory support.In addition to the scientific evidence suggesting the need to restore robust IFN responses, a phase II study conducted early in the course of the pandemic in patients hospitalised with COVID-19 showed that those who received SNG001 were more likely to improve, and recovered more rapidly, than those who received placebo . This prvia vibrating mesh nebuliser  for 14 days. The effect of study treatment was assessed on top of current local standard of care (SoC), with no limitation on concomitant medications for the treatment of COVID-19 or vaccination. Patients discharged during the 14-day treatment phase completed treatment at home, with study staff providing support via telephone or video call. Patients were followed for up to 90 days.In this double-blind, placebo-controlled, international study, patients were randomly assigned to SNG001 or matching placebo once daily ISRCTN registry (ISRCTN85436698). The protocol was amended four times (supplementary table S1).The study was approved by the independent ethics committees or research boards at each institution, performed in accordance with the principles of the Declaration of Helsinki and the International Conference on Harmonization notes for guidance on Good Clinical Practice (ICH/CPMP/135/95), and registered in the via nasal prongs or mask. Key exclusion criteria were ongoing SARS-CoV-2 infection that had lasted ≥3 weeks, previous SARS-CoV-2 infection, noninvasive ventilation, high-flow nasal oxygen therapy, endotracheal intubation and invasive mechanical ventilation . Patients provided informed consent prior to any study-related procedure.The study recruited males or females, ≥18 years of age, hospitalised due to COVID-19 and requiring oxygen −1 IFN-β1a). SNG001 dose selection was based on prior clinical and animal data . During nebulisation, supplemental oxygen could be administered via nasal cannula; if this was insufficient, additional oxygen could be administered through the nebuliser's oxygen port.Patients were assigned to treatment according to a randomisation schedule, with investigators and patients blinded to treatment by matching placebo. Study medication was presented in two pre-filled syringes, each containing 0.65 mL solution  Ordinal Scale of Clinical Improvement (OSCI) , BreathlThe primary objective was to evaluate recovery after administration of SNG001 compared with placebo. The two primary end-points were time to hospital discharge  and time to recovery to no limitation of activity . The study was to be considered successful if SNG001 was statistically superior to placebo for at least one of the primary end-points. The three key secondary end-points were: progression to severe disease or death (WHO OSCI score ≥5); progression to intubation or death (WHO OSCI score ≥6); and death, all assessed up to 35 days after first dose.Other secondary end-points were: the proportion of patients recovering , discharged from hospital and with an improvement in WHO OSCI, each at days 7, 14, 21 and 28; changes in BCSS total score and individual domains during the treatment period; changes in NEWS2 during the hospitalisation period; daily assessment of COVID-19 symptoms; limitation of usual activities; and quality of life measured using EQ-5D-5L. Safety and tolerability were assessed throughout the study by recording vital signs, adverse events, concomitant medications and immunogenicity.A sample size of 610 patients (n=305 per treatment arm) was estimated to provide ≥90% power to detect a hazard ratio (HR) of 1.45 in time to hospital discharge and a HR of 1.70 in time to recovery, with ≥95% power to declare statistical significance on at least one of the primary end-points. This sample size was calculated using a global two-sided a level of 0.05, adjusted with the Hochberg procedure to allow for multiple comparisons. The sample size calculation assumed 70% hospital discharge in the placebo arm at day 28, 30% recovery in the placebo arm at day 28 and a dropout rate of 25% over the 28-day evaluation period, with time to dropout exponentially distributed.supplementary material for the other secondary end-points.The hazard ratios for the two primary end-points were estimated from Cox proportional hazards models with covariates for age, sex, prior duration of COVID-19 symptoms, geographic region and COVID-19 vaccination status, with multiplicity controlled by the Hochberg procedure. For the key secondary end-points, odds ratios were estimated using logistic regression models with the same covariates as the primary analyses. See the supplementary table S2). The safety population consisted of the ITT population that received at least one dose of study medication.The intention-to-treat (ITT) population, used for the efficacy analyses, comprised all randomised patients. The per-protocol population, used for supportive analyses of the primary and key secondary end-points, comprised all patients in the ITT population who did not have any protocol deviations with an impact on efficacy (see Results and supplementary table S2). Baseline demographics and disease characteristics were similar in the two groups  days with SNG001 plus SoC compared with 8.0 (95% CI 7.0–9.0) days with placebo plus SoC, with a nonsignificant HR of 1.06 (95% CI 0.89–1.27); p=0.51. Results were similar in the per-protocol population (HR 1.02 (95% CI 0.84–1.23); p=0.85). The median time to recovery to no limitation of activity in the ITT population was 25.0  days in both treatment groups, with a nonsignificant HR of 1.02 (95% CI 0.81–1.28); p=0.89. Again, per-protocol population results were similar (HR 1.01 (95% CI 0.79–1.29); p=0.93).versus placebo plus SoC differences for the key secondary end-points were not statistically significant. The proportion of patients who progressed to severe disease or death by day 35 was 25.7% lower (OR 0.71 (95% CI 0.44–1.15); p=0.161) in the SNG001 plus SoC group compared with the placebo plus SoC group in the ITT population and 36.0% lower (OR 0.63 (95% CI 0.35–1.13); p=0.119) in the per-protocol population .SNG001 plus SoC pulation . Similarpost hoc analysis of progression to severe disease or death within 35 days (WHO OSCI score ≥5) was conducted, with patients subgrouped by baseline parameters that are associated with an increased risk of severe COVID-19: increased age (≥65 years), ≥1 comorbidities and poor respiratory function . This analysis was conducted in the per-protocol population so as to focus on the patients who had received study medication and clinical care according to the protocol stipulations. The odds ratios were higher in all subgroups compared with the overall per-protocol population, especially in patients with poor respiratory function in whom a significant  reduction was observed  to 69.9% (p=0.046) when patients were grouped by age, presence of comorbidities and poor respiratory function . This potential clinically important effect therefore needs to be confirmed in a future study adequately powered to assess this end-point.Efficacy analyses were also performed in the per-protocol population, which excluded patients whose treatment deviated from the protocol in a way that may have impacted evaluations. The most common reason for exclusion was not receiving at least two full doses of study medication in the first 3 days. The relative risk reduction in the patients who progressed to severe disease or death was 36% in the per-protocol population rather than 26% in the ITT population, although this was not statistically significant. Furthermore, in the i.e. matching the population recruited into SPRINTER) was 9 days in the placebo plus SoC group (data on file), it was 8 days in SPRINTER overall, decreasing further to 6 days in the UK sites . Improvements in SoC have also been reported by the RECOVERY Collaborative Group. Initially in the RECOVERY platform study, conducted in 2020, 28-day mortality was 23% in patients who received dexamethasone plus SoC [The lack of impact on recovery contrasts with the results from the phase II study of inhaled IFN-β in patients with COVID-19 , conductplus SoC , whereasplus SoC , 28.The favourable safety and tolerability profile of SNG001 observed in the SPRINTER study was consistent with the previous studies in patients with COVID-19, asthma and COPD , 23. A sGiven the observation, which would need to be confirmed in further studies, that patients with poor respiratory function may gain greater benefit from SNG001, a potential limitation of the study is that patients requiring noninvasive ventilation, high-flow nasal oxygen therapy, endotracheal intubation or invasive mechanical ventilation could not be dosed. However, the nebuliser can be used in different configurations that should enable these patients to be dosed in future studies . In addition, the timing of initiation of IFN treatment has been the subject of debate, with suggestions that later initiation could be less effective. Patients were excluded from the study only if the prior duration of symptoms was ≥3 weeks . In the previous phase II study, in which SNG001 was more effective than placebo, the median duration of symptoms at recruitment was similar to the current study . This suIn conclusion, although the primary objective of the study was not met, there were signals in the key secondary end-points which suggest that SNG001, on top of SoC, may have prevented progression to severe disease . In addition, SNG001 was well tolerated with a favourable safety profile, validating the route of administration. When combined with the results of the previous phase II study, these findings provide a rationale to continue investigating SNG001, not only in hospitalised patients with COVID-19 (in the context of ongoing virus evolution and likely emergence of new variants), but also more widely in patients with severe seasonal viral lung infections, due to the broad spectrum and variant agnostic antiviral activity of IFN-β.10.1183/23120541.00605-2022.Supp1Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.00605-2022.supplementSupplementary material